Acid phosphatases in serum can be separated by polyacrylamide gel electrophoresis into six bands, bands 0 to 5, from the starting point. Of these bands, band 5 shows resistance to tartrate, and is called band 5 tartrate-resistant acid phosphatase (TRACP5: tartrate-resistant acid phosphatase 5).
Band 5 is further separated into two bands by acidic disc electrophoresis, called TRACP5a and TRACP5b, respectively. TRACP5a is an enzyme originating from platelets and other components, and its blood level does not change. On the other hand, TRACP5b is considered to derive from osteoclasts, since its blood level changes with bone resorption. TRACP5b is needed in large amounts to screen for candidate compounds in developing TRACP5b-specific inhibitors, activators, or modulators.
Therefore, to date, many researchers have attempted to produce useful human TRACP5b. However, there are no successful examples of expressing TRACP5b directly in vivo, and it had to be purified from large amounts of osteoclastoma (Non-patent Document 1), or isolated from large amounts of fresh serum (Non-patent Document 2). Meanwhile, attempts to produce TRACP5b by expressing TRACP5 in insect cells (Non-patent Document 3) and allowing protease to act on the obtained TRACP5 in vitro, are known. However, the fact is that only a small amount of the desired substance can be obtained (Non-patent Document 4).
Previously, the present inventors have made attempts to produce TRACP5b by conducting various modifications on Escherichia coli, insect cells, mammalian cells, and such, but they were unsuccessful. The reason for this was that the special post-translational modification (Non-patent Document 4) during the production of TRACP5b cannot be reproduced in these systems. The present inventors also produced TRACP5 and then treated it with enzymes such as cathepsin K in vitro, but failed to establish a production method for an enzyme protein that is stable from the viewpoint of industrialization.
In recent years, techniques for producing gene products using silkworms have been developed, and research on methods for introducing foreign genes and regulating the expression of transgenes has progressed. They are expected to become novel methods for protein production. For example, it is known that recombinant proteins are produced by expressing transgenes in the silk gland using transgenic silkworm production technology. Since silkworms are eukaryotic organisms, they can produce proteins close to mammalian types, as compared to plants or microorganisms such as E. coli. In addition, since silkworms have silk glands, which are organs suitable for producing recombinant proteins, they are capable of producing almost 1 g of protein per animal. Furthermore, silkworms can be reared under clean conditions using an artificial diet, and large-scale rearing at the level of several tens of thousands of silkworms can be easily carried out. In addition, by carrying out the large-scale rearing of silkworms using mulberry leaves abundant in nature, large amounts of recombinant proteins can be obtained at a low cost.
Meanwhile, there are several known methods for producing virus-infected silkworm larvae using recombinant baculoviruses, and obtaining useful proteins such as interferons which are used for pharmaceuticals (antitumor/antiviral agents or such) and the like, from their body fluid. It can be said that these methods have also provided novel protein production methods that utilize the ability of silkworms to produce proteins.    [Non-patent Document 1] J. J. Stepan, K. H. W. Lau, S. Mohan, F. R. Singer, D. J. Baylink, (1990) Purification and N-Terminal Acid Sequence of the Tartrate-Resistant Acid Phosphatase from Human Osteoclastoma Evidence for a Single Structure. Biochemical and Biophysical Research Communications 168, 792-800.    [Non-patent Document 2] K. W. Lam, D. Ted Eastlund, Chin-Yang Li, Lung T. Yam, (1978) Biochemical Properties of Tartrate-Resistant Acid Phosphatase in Serum of Adults and Children Clinical Chemistry 24, 1105-1108.    [Non-patent Document 3] Alison R. Hayman, Timotyh M. Cox, (1994) Purple Acid Phosphatase of the Human Macrophage and Osteoclast. The Journal of Biological Chemistry 269, 1294-1300.    [Non-patent Document 4] Jenny Ljusberg, Yunling Wang, Pernilla Lang, Maria Norgard, Robert Dodds, Kjell Hultenby, Barbro Ek-Rylander, Goran Andersson, (2005) Proteolytic Excision of a Repressive Loop Domain in Tartrate-resistant Acid Phosphatase by Cathepsin Kin Osteoclasts. The Journal of Biological Chemistry 280, 28370-28381.